Axiocam 506 mono ccd

Infected cells were fixed with 4% paraformaldehyde (PFA) in PBS (Nzytech) for 15 min at room temperature. Fixed cells were permeabilized with 0.2% Saponin (Sigma) in PBS containing 2% BSA (Nzytech), for 30 min at room temperature. Transfected cells were fixed with 4% PFA at room temperature for 10 min, permeabilized with 0.3% Triton X-100 in PBS for 5 min and blocked with 5% BSA in PBS for 1 h at room temperature. For immunostaining, coverslips were incubated with primary antibodies diluted in blocking solution (2 h, room temperature), washed with PBS, incubated for 1 h at room temperature with Alexa fluor-conjugated secondary antibodies (Molecular probes, Invitrogen, Thermo Scientific) and Hoechst 33342 (Invitrogen) and washed again. The coverslips were mounted on microscope slides with Fluoromount G (Invitrogen). For infection quantification by microscopy, parasites were stained with anti-PbHSP70(Tsuji et al., 1994 (link)) or anti-GFP Alexa fluor-488 conjugate (Invitrogen) and imaged on a wide-field microscope equipped with an automated stage (Zeiss Axio observer, 40x Air (0.75), EC Plan-NeoFluar, Axiocam 506 mono-CCD (4.54∗4.54 μm pixel size)). High-resolution images were acquired on point scanning confocal microscopes (Zeiss LSM 880 or LSM 710, 63x Oil (1.04) Plan-Apochromat DIC). All images were processed and analysed using ImageJ/FIJI (Schindelin et al., 2012 (link)).